Without the development of new therapeutic antimicrobials, it is estimated that AMR will cause 10 million annual deaths by 2050. Lanthipeptides possess a range of therapeutic properties, including antimicrobial activities, and could potentially be developed into therapeutic antimicrobials. Lanthipeptides are ribosomally synthesised post translationally modified peptides characterised by cyclic thioether Lanthionine (Lan) rings. Lan rings provide lanthipeptides with conformational rigidity that contributes towards not only to their bioactivity, but to their potency, stability, and specificity. A recently discovered lanthipeptide, lexapeptide, has Lan rings installed by a newly discovered fifth class of lanthipeptide synthetases. Lexapeptide has potent antimicrobial activities against pathogenic bacteria such as MRSA and has potential to be developed into a new therapeutic antimicrobial. Thus, we will investigate how Lan is installed by Class V lanthipeptide synthetases in lexapeptide.
Here, we show that the Lan moiety in lexapeptide is installed by a set of three enzymes, LxmK, LxmY, and LxmX, which are unique to known lanthipeptide synthetases. Our crystal structure of the LxmKY complex indicates that the kinase (LxmK) and lyase (LxmY), respectively, phosphorylates and dephosphorylates Ser/Thr residues to generate the corresponding dehydrated amino acids. Our crystal structure of LxmX suggests a novel putative cyclase that catalyses the formation of the Lan thioether ring between cysteine and the dehydrated residues. The biochemical activity of these enzymes are established using synthetic peptides and tandem mass spectrometry, confirming the roles these enzymes play in lexapeptide biosynthesis. Our results elucidate how Class V lanthipeptide synthetases install Lan in lexapeptide by a cooperative system of three enzymes. This enables future mechanistic work to be performed that will further deepen our knowledge and lay the foundation to developing lexapeptide as a therapeutic antimicrobial.